Masaharu Hisasue

Analysis of the Disease Potential of a Recombinant Retrovirus Containing Friend Murine Leukemia Virus Sequences and a Unique Long Terminal Repeat from Feline Leukemia Virus

ABSTRACT We have molecularly cloned a feline leukemia virus (FeLV) (clone 33) from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV proviruses from other cats with AML, contains an unusual structure in its U3 region upstream of the enhancer (URE) consisting of three tandem direct repeats of 47 bp. To test the disease potential and specificity of this unique FeLV LTR, we replaced the U3 region of the LTR of the erythroleukemia-inducing Friend murine leukemia virus (F-MuLV) with that of FeLV clone 33. When the resulting virus, F33V, was injected into newborn mice, almost all of the mice eventually developed hematopoietic malignancies, with a significant percentage being in the myeloid lineage. This is in contrast to mice injected with an F-MuLV recombinant containing the U3 region of another FeLV that lacks repetitive URE sequences, none of which developed myeloid malignancies. Examination of tumor proviruses from F33V-infected mice failed to detect any changes in FeLV U3 sequences other than that in the URE. Like F-MuLV-infected mice, those infected with the F-MuLV/FeLV recombinants were able to generate and replicate mink cell focus-inducing viruses. Our studies are consistent with the idea that the presence of repetitive sequences upstream of the enhancer in the LTR of FeLV may favor the activation of this promoter in myeloid cells and contribute to the development of malignancies in this hematopoietic lineage.

Myelodysplastic syndromes and acute myeloid leukemia in cats infected with feline leukemia virus clone33 containing a unique long terminal repeat.

Feline leukemia virus (FeLV) clone33 was obtained from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV from other cats with AML, differs from the LTRs of other known FeLV in that it has 3 tandem direct 47‐bp repeats in the upstream region of the enhancer (URE). Here, we injected cats with FeLV clone33 and found 41% developed myelodysplastic syndromes (MDS) characterized by peripheral blood cytopenias and dysplastic changes in the bone marrow. Some of the cats with MDS eventually developed AML. The bone marrow of the majority of cats with FeLV clone33 induced MDS produced fewer erythroid and myeloid colonies upon being cultured with erythropoietin or granulocyte‐macrophage colony‐stimulating factor (GM‐SCF) than bone marrow from normal control cats. Furthermore, the bone marrow of some of the cats expressed high‐levels of the apoptosis‐related genes TNF‐α and survivin. Analysis of the proviral sequences obtained from 13 cats with naturally occurring MDS reveal they also bear the characteristic URE repeats seen in the LTR of FeLV clone33 and other proviruses from cats with AML. Deletions and mutations within the enhancer elements are frequently observed in naturally occurring MDS as well as AML. These results suggest that FeLV variants that bear URE repeats in their LTR strongly associate with the induction of both MDS and AML in cats.

Prothrombotic and Inflammatory Effects of Intravenous Administration of Human Immunoglobulin G in Dogs

BACKGROUND Intravenous administration of human immunoglobulin G (hIVIgG) has been suggested to potentiate thromboembolism in dogs, but supportive scientific reports are lacking. OBJECTIVES To determine if hIVIgG therapy promotes hypercoagulability and inflammation in dogs. ANIMALS Twelve healthy Beagle dogs. METHODS Prospective, experimental trial. An hIVIgG/saline solution was infused IV at 1 g/kg BW over 8 hours to 6 dogs, and physiological saline was infused to the other 6 dogs. Blood samples were drawn before, during, and after infusion for serial measurement of indicators of coagulation and inflammation. Data were analyzed by 2-way repeated measures analysis of variance. RESULTS Dogs administered hIVIgG developed mildly decreased blood platelet concentrations without thrombocytopenia (median, 200 x 10(3)/microL; range, 150-302 x 10(3)/microL; P < .01), leukopenia (median, 3.5 x 10(3)/microL; range, 20-62 x 10(3)/microL; P < .001), and mildly increased plasma total protein concentrations (median, 6.3 g/dL; range, 5.6-6.7 g/dL; P < .001). Administration of hIVIgG was also associated with increases in fibrin/fibrinogen degradation products in all dogs (either 5 microg/mL or 10 microg/dL), thrombin-antithrombin III complexes (median, 7.2 ng/mL; range, 4.9-14.2 ng/mL; P < .001), and C-reactive protein concentrations (median, 2.5 mg/dL; range, 0.5-4.3 mg/dL; P < .01). CONCLUSION AND CLINICAL IMPORTANCE Administration of hIVIgG to dogs promotes hypercoagulability and an inflammatory state. This should be further evaluated and considered when using hIVIgG in dogs with IMHA or other prothrombotic conditions.

Canine bone marrow cells differentiate into hepatocyte-like cells and placental hydrolysate is a potential inducer

Hepatocyte growth factor (HGF) can stimulate human and rat bone marrow (BM) cells to differentiate into hepatocytes. A human placental hydrolysate (hPH) stimulates proliferation of hepatocytes, but its role as a potential inducer of BM cells to form hepatocytes is unclear. To determine if canine BM cells stimulated with HGF or hPH differentiate into hepatocyte-like cells, BM cells were cultured with HGF or hPH. The cultured cells underwent morphological examination, expression of albumin and cytokeratin 18 (CK18), hepatic function tests including uptake of low-density lipoprotein (LDL) and cytochrome P (CYP) 450 activity. Albumin mRNA and protein expression of albumin and CK18 proteins were detected in cultures with HGF and hPH. Furthermore, these cells demonstrated LDL uptake and CYP450 activity. These results indicate that canine BM cells can differentiate into hepatocyte-like cells when stimulated by both HGF and that hPH may be an effective inducer of hepatic differentiation.

Serological evidence of infection of dogs with human influenza viruses in Japan

Historically, influenza virus has not been regarded as a major pathogen of dogs. However, recent infections of racing and pet dogs with H3N8 virus of equine origin in the USA after 2004 (Crawford and others 2005) and retrospectively in the UK in 2002 (Daly and others 2008), as well as with highly pathogenic H5N1 avian virus in Thailand in 2004 (Songserm and others 2006), revealed that dogs are susceptible to influenza A viruses. These infections caused respiratory disease in the dogs and several proved fatal. Moreover, H3N2 virus of avian origin infected pet dogs in Korea in 2007 (Song and others 2008) and China in 2010 (Li and others 2010), supporting the belief that dogs should be included among the animals that are responsible for interspecies transmission of influenza A virus (Kim and others 2013). Furthermore, there were reports in various parts of the world (Dundon and others 2010 …

Bone marrow colony-forming unit assay in cats with naturally occurring myelodysplastic syndromes

Feline myelodysplastic syndromes (MDS) has been diagnosed in many cats infected with feline leukemia virus, although the pathogenesis of this hematopoietic deficiency has been unclear. In this study, we assayed the bone marrow erythroid colony-forming units (CFU-E) and granulocyte-machrophage CFUs (CFU-GM) to investigate the pathogenesis of feline MDS. The number of CFU-E colonies was decreased in 4 of 7 cats with MDS, and the number of CFU-GM colonies was also decreased in 4 cats. Furthermore, small colonies of CFU-GM were found in all 7 cases. These findings indicated that refractory cytopenia of feline MDS could be caused by abnormal maturation and differentiation of hematopoietic stem cells in bone marrow, as it is in human MDS. The pathogenesis of feline MDS might be similar to that of human MDS.

Valproic acid, a histone deacetylase inhibitor, decreases proliferation of and induces specific neurogenic differentiation of canine adipose tissue-derived stem cells.

ABSTRACT Adipose tissue-derived stem cells (ADSCs) isolated from adult tissue have pluripotent differentiation and self-renewal capability. The tissue source of ADSCs can be obtained in large quantities and with low risks, thus highlighting the advantages of ADSCs in clinical applications. Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to affect ADSC differentiation in mice and rats; however, few studies have been performed on dogs. We aimed to examine the in vitro effect of VPA on canine ADSCs. Three days of pretreatment with VPA decreased the proliferation of ADSCs in a dose-dependent manner; VPA concentrations of 4 mM and above inhibited the proliferation of ADSCs. In parallel, VPA increased p16 and p21 mRNA expression, suggesting that VPA attenuated the proliferative activity of ADSCs by activating p16 and p21. Furthermore, the effects of VPA on adipogenic, osteogenic or neurogenic differentiation were investigated morphologically. VPA pretreatment markedly promoted neurogenic differentiation, but suppressed the accumulation of lipid droplets and calcium depositions. These modifications of ADSCs by VPA were associated with a particular gene expression profile, viz., an increase in neuronal markers, that is, NSE, TUBB3 and MAP2, a decrease in the adipogenic marker, LPL, but no changes in osteogenic markers, as estimated by reverse transcription-PCR analysis. These results suggested that VPA is a specific inducer of neurogenic differentiation of canine ADSCs and is a useful tool for studying the interaction between chromatin structure and cell fate determination.

Cats as a potential source of emerging influenza virus infections.

Based on the findings of the present study, we conclude that cats can be infected with human influenza viruses as well as avian influenza viruses. Actually, the recent study has shown that both human-type(α2, 6-linked sialic acid) and avian-type(α2, 3-linked sialic acid)influenza virus receptors were extensively detected in the respiratory organs such as trachea, bronchus, and lung of the domestic cats(Wang et al., 2013). Therefore, cats may act as a vector for human influenza virus transmission within households, posing a potential public health concern. Furthermore, we detected both H5N1 and human virus-seropositive cats in neighboring areas at similar sampling times, suggesting that cats can be simultaneously infected with both avian and human viruses in H5N1 virus-endemic areas. Thus, cats, like pigs, may act as an intermediate host for the emergence of new, potentially p and emic viruses.

Effects of Prostaglandin E1 on the Preparation of Platelet Concentrates in Dogs

BACKGROUND Platelet concentrates (PC) are prepared by centrifugation of platelet-rich plasma (PRP) that is prepared by centrifugation of whole blood. The resuspension of the platelet pellet during PC preparation from dogs is difficult because of platelet activation induced by centrifugation. OBJECTIVES To investigate the efficacy of adding prostaglandin E(1) (PGE(1) ) to prevent platelet activation during PC preparation from dogs. ANIMALS Fifteen healthy Beagle dogs. METHODS Prospective, experimental trial: PGE(1) was added to PRP before the high-speed centrifugation during PC preparation. To estimate the effect of this addition, we assessed the platelet aggregability before transfusion, the survival of the platelets after transfusion, and the platelet reactivity after transfusion, which is estimated by the P-selectin expression of the platelets when stimulated by thrombin. RESULTS The difficulty associated with platelet resuspension was resolved by PGE(1.) PGE(1) strongly inhibited platelet aggregation induced by collagen and ADP; however, it recovered after the platelets were resuspended in plasma without PGE(1) (mean aggregation ratio; collagen: 10.00-80.80%, ADP: 8.20-53.60%). Survival of the platelets after transfusion was not affected by PGE(1) (mean 8.04 and 7.56 days, without and with PGE(1) ), and thrombin-induced P-selectin expression after transfusion in PGE(1) -treated PC was also well maintained (mean positive ratio 53.7 and 47.9%, before and 24 hours after transfusion). CONCLUSIONS AND CLINICAL IMPORTANCE The addition of PGE(1) in PRP before the centrifugation of PRP can improve the preparation efficiency of PC from dogs, while maintaining the therapeutic efficacy of the platelets.

AKT capture by feline leukemia virus

Oncogene-containing retroviruses are generated by recombination events between viral and cellular sequences, a phenomenon called “oncogene capture”. The captured cellular genes, referred to as “v-onc” genes, then acquire new oncogenic properties. We report a novel feline leukemia virus (FeLV), designated “FeLV–AKT”, that has captured feline c-AKT1 in feline lymphoma. FeLV–AKT contains a gag–AKT fusion gene that encodes the myristoylated Gag matrix protein and the kinase domain of feline c-AKT1, but not its pleckstrin homology domain. Therefore, it differs structurally from the v-Akt gene of murine retrovirus AKT8. AKT may be involved in the mechanisms underlying malignant diseases in cats.